Cryo-replicator The cryo-replicator CR1000 has 96 spring-loaded pins and allows the simultaneous sampling of 96 frozen glycerol stocks (-80 ºC) without thawing the remaining glycerol culture. Since the "mother-cultures" remain frozen, their viability (number of CFU's sampled) is not affected by repeated sampling. Per sampling, less than 0.5 µl is removed from each stock culture so that one master plate can be used for several hundreds of times. The pins are 45 mm long and can also be used for the purpose of transferring 96 colonies simultaneously into a deepwell plate containing liquid growth medium. Note: also replicators with 24 pins are available (ask)

Background information: The cryo-replicator samples frozen stocks not by scratching material from the surface (as is current practice when sampling an individual frozen stock with a needle), but by means of heat transfer instead. The amount of heat transferred from the individual pins (at room temperature) to the -80 ºC cultures in a contact period of 3 seconds is just sufficient to melt 0.3 µl, which forms a thin film at the end of the pins. Since the film of sampled cells on the pins has a thickness of only 0.05 mm, cross-contamination by e.g. falling droplets does not occur. All stainless steel pins (thickness 3 mm) are individually sprung with light stainless steel springs to allow all 96 cultures to be sampled simultaneously. Since the pins can move up and down under the back-pressure of springs, it is not necessary that the frozen surfaces of all 96 cultures are exactly at an equal level (which is practically impossible anyway, see note 1 below). When the cryo-replicator is used (and the master-plate is always placed back at -80 ºC immediately after use), a single same master plate can be used for years and hundreds of samplings. As stated above, when using the cryo-replicator for sampling master-plates, the exact volumes in the individual wells of the master plate have no influence on the amounts of cell cultures sampled. This situation is also helpful for an easy 'maintenance' of master plates; if one of the strains from a heterogeneous culture collection has lost its viability (e.g. some Pseudomonas putida strains are prone to lose viability at -80 ºC within a year), a fresh glycerol stock culture can be added on top of the old (dead) cells of the well. The surface in this particular well is now a few mm higher than the others, but this will pose no problems during sampling. The sole effect is that the pin corresponding to this culture will move up a bit further than the others (see the photos at the left) The replicator is sterilised by placing it on a hot plate (250-300 ºC) for 5 minutes. The subsequent cooling down to room temperature requires 12 minutes. For an efficient use of time, it is advisable to work alternately with three cryo-replicators: one can work with one replicator when the other two are being sterilised or are cooling down. Warning: the cryo-replicator should not be autoclaved. Autoclaving can cause serious damage to the cryo-replicator. Sterilisation is achieved via use of a hot plate (see manual).

Schematic view of the sampling of a -80 ºC master-plate filled with glycerol cultures note 1: Preparation of a frozen master plate that has identical surface levels is pratically impossible: · due to the high viscosity of cell suspensions in a glycerol solution, pipetting errors of 10% are common · during freezing, the surface of some cultures may become hollow, or dome-shaped · At -80 ºC, plastic tends to deform to some extent: polypropylene or polystyrene microtiter plates used as master plates do not remain completely flat at -80 ºC The alternative (using a regular fixed-pin replicator) has the consequence that the master-plate needs to be (partially or fully) melted before sampling. Repeated freeze-thaw cycles result in a rapid decrease in the viability of the stored cells. For e.g. E. coli cells, 4 or 5 freeze-thaw cycles generally result in a loss of viability of more than 99.9%